Method for determination of cholinesterase activity

Abstract

In the method according to the present invention and with the use of reagents according to the present invention, it has been possible to perform routine examinations of activities of cholinesterase in serum, that is of high importance for examinations of liver function disease. A kinetic determination method with using an autoanalyzer can be performed in reacting a test sample of serum with p-hydroxybenzoyl choline as a substrate in presence of p-hydroxybenzoic acid hydroxylase.

We claim: 
     
       1. The method for determination of cholinesterase activity by measuring the extinction decrease or the oxygen consumption, comprising the following steps: (a) Subjecting a substrate having the formula: ##STR3##  wherein R 1  and R 2  represent a hydrogen atom or one or both of R 1  and R 2  represent a lower-alkoxy group with 1 through 4 carbon atoms, and X represents a halogen atom, to hydrolysis by the action of cholinesterase to produce a p-hydroxybenzoic acid,   (b) reacting the p-hydroxybenzoic acid thus-produced with p-hydroxybenzoic acid hydroxylase in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate in reduced form (NADPH), whereby the NADPH is oxidized to nicotinamide adenine dinucleotide phosphate (NADP), and   (c) measuring the extent of extinction decrease or oxygen consumption in the reaction mixture produced by conversion of the nicotinamide-adenine-dinucleotide phosphate in reduced form (NADPH) to the oxidized form (NADP).   
     
     
       2. Method according to claim 1 characterized in that the extinction is measured at 340 nm. 
     
     
       3. Method according to claim 1 characterized in that the speed of oxygen consumption in the reaction solution is measured by means of an oxygen electrode. 
     
     
       4. Method according to claim 1 characterized in that the reaction of p-hydroxy-benzoic-acid-hydroxylase is performed in the presence of flavin adenine dinucleotide (FAD). 
     
     
       5. Method according to claim 1 characterized in that the reaction of cholinesterase with substrate is performed at a pH value of 7.5 to 9.5. 
     
     
       6. Method according to claim 5 characterized in that the reaction is performed in the presence of a buffer. 
     
     
       7. Method according to claim 6 wherein the pH is about 8.2. 
     
     
       8. Reagent for determination of cholinesterase activity consisting essentially of a halide salt of p-hydroxybenzoylcholine optionally having one or two lower(1-4 C atom)alkoxy groups on the phenyl ring in position adjacent to the p-hydroxy group, p-hydroxybenzoic acid hydroxylase, and coenzyme nicotinamide-adenine-dinucleotide-phosphate in reduced form (NADPH), and one or more additional ingredients selected from the group consisting of FAD, salicyclic acid, and buffer, the proportions of p-hydroxybenzoylcholine, p-hydroxybenzoic acid hydroxylase, and NADPH being such that, upon subjection of the reagent to cholinesterase, the p-hydroxybenzoylcholine converts to a benzoic acid, which reacts with the p-hydroxybenzoic acid hydroxylase to give a measurable oxidation of the NADPH to NADP. 
     
     
       9. Reagent according to claim 8 characterized in that it contains 0.02 to 20 mM p-hydroxybenzoyl choline, 0.1 to 0.7 mM nicotinamide-adenine-dinucleotide phosphate in reduced form, 200 to 5,000 U/l p-hydroxybenzoic acid hydroxylase, and 0.1 to 30 μM FAD buffered to pH 7.5 to 9.5. 
     
     
       10. Reagent according to claim 9 characterized in that buffer is tris-(hydroxymethyl)-aminomethane-maleic acid. 
     
     
       11. A method for determining the activity of cholinesterase ##STR4##  wherein R 1  and R 2  represent a hydrogen atom or one or both of R 1  and R 2  represent a lower alkoxy group with 1 to 4 carbon atoms under conditions effective to transform said substrate into p-hydroxybenzoic acid and to oxidize said NADPH to NADP+ and measuring the decrease in extinction produced by the conversion of NADPH to NADP+.